Figure 3 - uploaded by Rustem R Islamov
Content may be subject to copyright.
Confocal immunofluorescence for p100, AGO2, Gemin3, and FMRP proteins in sciatic nerve fibers. Longitudinal sections of sciatic nerves were incubated with primary antibodies against p100, AGO2, and Gemin3 or FMRP and subsequently incubated with secondary antibodies conjugated with Texas Red, FITC, or CY5. All primary antibodies were applied in a concentration of 1:100. Upper panel shows triple staining against p100 (blue), AGO2 (red), and Gemin3 (green). Lower panel shows triple staining against p100 (blue), AGO2 (red), and FMRP (green). The fluorescent signals indicate that the major components of RISC (p100, AGO2, Gemin3, and FMRP) are expressed in sciatic nerve fibers (indicated by white arrows).
Source publication
Recent observations demonstrated that translation of mRNAs may occur in axonal processes at sites that are long distances away from the neuronal perikaria. While axonal protein synthesis has been documented in several studies, the mechanism of its regulation remains unclear. The aim of this study was to investigate whether RNA interference (RNAi) m...
Contexts in source publication
Context 1
... aim of this experiment was to determine whether the known components of mammalian RISC (AGO2, FMRP, and p100) were present in the sciatic nerve fibers. For these experiments we chose to target neuronal  -tubulin because it is one of the abundant cytoskeletal proteins known to be synthesized locally in the axonal fibers (38). Proximal stumps of sciatic nerves transected at midthigh level were incubated with antitubulin siRNA, nonspecific control siRNA, or vehicle for 24 h. For protein extraction we used a 2 cm portion of sciatic nerve 0.5 cm above the site of treatment ( Fig. 1). For spinal cord protein extracts we used lumbar spinal cord from the corresponding animals. Protein samples isolated from sciatic nerves and spinal cords were resolved by SDS/PAGE electrophoresis and transferred to membranes, which were subsequently probed with antibodies against AGO2, FMRP, and p100. These antibodies have been successfully used elsewhere (14). Immunoblot analysis revealed the presence of AGO2, FMRP, and p100 proteins in samples from the spinal cord ( Fig. 2 , upper panel) and sciatic nerves (Fig. 2, lower panel). In sciatic nerves, pretreatment with antitubulin siRNA visibly increased the level of p100 expression, which may be a sign of induction by siRNA. The data of this experiment demonstrated that the three known components of RISC (AGO2, FMRP, and p100) were present in sciatic nerve fibers. To confirm our finding at the histological level, we performed immunofluorescence on sciatic nerve fibers to investigate the distribution of the known (AGO2, FMRP, and p100) and suspected (Gemin3) components of RISC. Longitudinal sections of an ϳ 2 cm portion of sciatic nerve proximal stump ( ϳ 0.5 cm above the site of siRNA treatment) were incubated with primary antibodies against target proteins and subsequently incubated with secondary antibodies conjugated with Texas Red, FITC, or CY3. The sections were analyzed using a Zeiss LSM 510 confocal laser scanning microscope. Triple immunostaining of these sections revealed immunoreactivity in the sciatic nerve for p100 (blue color-coded), AGO2 (red), FMRP (green), and Gemin3 (green) proteins ( Fig. 3 , upper and lower panels). A merge of the images showed coexpression of p100 (blue), AGO2 (red), Gemin3 (green) (Fig. 3, upper panel) as well as coexpression of p100 (blue), AGO2 (red), and FMRP (green) (Fig. 3, lower panel). These data demonstrated that AGO2, FMRP, p100, and Gemin3 coex- press in peripheral nerve fibers, providing the neces- sary substrates for the formation of RISC. To investigate whether RISC proteins are present in axons of dissociated neuronal cultures, we performed immunofluorescence on rat DRG neurons plated at low density according to a protocol described elsewhere (37, 38). The preparations were stained with antibodies against AGO2, p100, FMRP, and Gemin3, as well as with TUJ1 antibodies against neuronal  -tubulin and antibodies against growth-associated protein 43 (GAP43). In some experiments, DRG cultures were treated with antitubulin siRNA or nonspecific control for 24 h before immunostaining. Immunofluorescence showed colocalization of p100 with  -tubulin ( Fig. 4 A ), p100 with FMRP (Fig. 4 B ), p100 with AGO2 (Fig. 4 C ), and p100 with Gemin3 (Fig. 4 D ). A merge of the images showed a complete overlap between immunofluorescence of the proteins of interest in axons as well as in cell bodies. Incubation of DRG cultures with siRNAs demonstrated successful uptake of siRNA duplexes. FITC-conjugated siRNAs were detected in axons and bodies of DRG neurons. Triple immunofluorescence of DRG cultures treated with antitubulin siRNA confirmed colocalization of p100 with  -tubulin (Fig. 4 E ), p100 with GAP43 (Fig. 4 F ), p100 with AGO2 (Fig. 4 G ), and p100 with FMRP (Fig. 4 H ). These results clearly demonstrated the existence of RISC proteins in axons. To confirm this finding at the protein level, we performed immunoblot analysis on axonal proteins isolated from DRG cultures according to a method described previously (38). This method allows the separation of DRG processes from cell body and non- neuronal cells by culturing neurons on a porous membrane that allows passage of axons but restricts the cell body and non-neuronal cells to the upper membrane surface. Proteins extracted from the cell body and axonal preparations were separated by 7.5% SDS-PAGE (10 g/lane) and transferred to PVDF membrane. To demonstrate the purity of the DRG axonal preparation, membranes were probed with antibodies against MAP-2 and GAP43. MAP2 protein resides in the cell soma and dendrites but does not extend into the axon (37), whereas GAP43 is localized to the growing axons and soma (56). Our immunoblot showed that MAP2 was present in the cell body fraction but absent from axonal preparation, whereas GAP43 was more abundant in the axonal preparation than in the cell body fraction, thus confirming the purity of the axonal fraction. The immunoblot analysis revealed the presence of RISC proteins (p100, AGO2, FMRP, and Gemin3) in both the cell body and axonal fractions ( Fig. 5 ). Together with the immunofluorescence experiments on sciatic nerve fibers and dissociated DRG cultures, these data argue that axons possess proteins specific for RISC. The purpose of this experiment was to determine whether AGO2, FMRP, p100, and Gemin3 can form protein complexes in sciatic nerve fibers in response to treatment with siRNA against neuronal  -tubulin. The proteins were isolated from ϳ 1 cm distal and ϳ 1 cm proximal parts of the proximal stump dissected out 0.5 cm above the site of siRNA application. The control treatments included saline and nonspecific siRNA. Protein lysates were incubated with AGO2 or p100 antibodies overnight and precipitated using BSA preblocked protein A-Sepharose beads. The precipitated proteins were separated according to relative size by SDS-PAGE analysis in 5% gels, transferred to membranes, and probed with antibodies against AGO2, FMRP, p100, and Gemin3. In all treatment conditions, p100 was shown to coprecipitate with FMRP and AGO2 ( Fig. 6 , upper panel), whereas AGO2 was shown to coprecipitate with p100 and Gemin3 (Fig. 6, middle panel). As expected, expression of the multiprotein complex was more pronounced in response to treatment by siRNA, indicating that the presence of siRNA induces RISC formation (14). To investigate whether RNAi may act independently from the neuronal cell body, experiments with ligature and colchicine treatment were performed. The ligature was used to mechanically separate the sciatic nerve proximal stump from the neuronal cell bodies. Colchicine, which causes depolymerization of microtubules (57), was used to block axonal transport pharmacolog- ically without affecting the nerve capacity to conduct action potentials. Ligation or colchicine were applied for 24 h at the proximal stump of the sciatic nerve ϳ 1.5 cm above the site of the treatment with siRNAs. Experiments showed that neither the ligature nor colchicine affected RISC protein levels in the proximal stump above or below the ligature/colchicine application site. This observation ...
Context 2
... aim of this experiment was to determine whether the known components of mammalian RISC (AGO2, FMRP, and p100) were present in the sciatic nerve fibers. For these experiments we chose to target neuronal  -tubulin because it is one of the abundant cytoskeletal proteins known to be synthesized locally in the axonal fibers (38). Proximal stumps of sciatic nerves transected at midthigh level were incubated with antitubulin siRNA, nonspecific control siRNA, or vehicle for 24 h. For protein extraction we used a 2 cm portion of sciatic nerve 0.5 cm above the site of treatment ( Fig. 1). For spinal cord protein extracts we used lumbar spinal cord from the corresponding animals. Protein samples isolated from sciatic nerves and spinal cords were resolved by SDS/PAGE electrophoresis and transferred to membranes, which were subsequently probed with antibodies against AGO2, FMRP, and p100. These antibodies have been successfully used elsewhere (14). Immunoblot analysis revealed the presence of AGO2, FMRP, and p100 proteins in samples from the spinal cord ( Fig. 2 , upper panel) and sciatic nerves (Fig. 2, lower panel). In sciatic nerves, pretreatment with antitubulin siRNA visibly increased the level of p100 expression, which may be a sign of induction by siRNA. The data of this experiment demonstrated that the three known components of RISC (AGO2, FMRP, and p100) were present in sciatic nerve fibers. To confirm our finding at the histological level, we performed immunofluorescence on sciatic nerve fibers to investigate the distribution of the known (AGO2, FMRP, and p100) and suspected (Gemin3) components of RISC. Longitudinal sections of an ϳ 2 cm portion of sciatic nerve proximal stump ( ϳ 0.5 cm above the site of siRNA treatment) were incubated with primary antibodies against target proteins and subsequently incubated with secondary antibodies conjugated with Texas Red, FITC, or CY3. The sections were analyzed using a Zeiss LSM 510 confocal laser scanning microscope. Triple immunostaining of these sections revealed immunoreactivity in the sciatic nerve for p100 (blue color-coded), AGO2 (red), FMRP (green), and Gemin3 (green) proteins ( Fig. 3 , upper and lower panels). A merge of the images showed coexpression of p100 (blue), AGO2 (red), Gemin3 (green) (Fig. 3, upper panel) as well as coexpression of p100 (blue), AGO2 (red), and FMRP (green) (Fig. 3, lower panel). These data demonstrated that AGO2, FMRP, p100, and Gemin3 coex- press in peripheral nerve fibers, providing the neces- sary substrates for the formation of RISC. To investigate whether RISC proteins are present in axons of dissociated neuronal cultures, we performed immunofluorescence on rat DRG neurons plated at low density according to a protocol described elsewhere (37, 38). The preparations were stained with antibodies against AGO2, p100, FMRP, and Gemin3, as well as with TUJ1 antibodies against neuronal  -tubulin and antibodies against growth-associated protein 43 (GAP43). In some experiments, DRG cultures were treated with antitubulin siRNA or nonspecific control for 24 h before immunostaining. Immunofluorescence showed colocalization of p100 with  -tubulin ( Fig. 4 A ), p100 with FMRP (Fig. 4 B ), p100 with AGO2 (Fig. 4 C ), and p100 with Gemin3 (Fig. 4 D ). A merge of the images showed a complete overlap between immunofluorescence of the proteins of interest in axons as well as in cell bodies. Incubation of DRG cultures with siRNAs demonstrated successful uptake of siRNA duplexes. FITC-conjugated siRNAs were detected in axons and bodies of DRG neurons. Triple immunofluorescence of DRG cultures treated with antitubulin siRNA confirmed colocalization of p100 with  -tubulin (Fig. 4 E ), p100 with GAP43 (Fig. 4 F ), p100 with AGO2 (Fig. 4 G ), and p100 with FMRP (Fig. 4 H ). These results clearly demonstrated the existence of RISC proteins in axons. To confirm this finding at the protein level, we performed immunoblot analysis on axonal proteins isolated from DRG cultures according to a method described previously (38). This method allows the separation of DRG processes from cell body and non- neuronal cells by culturing neurons on a porous membrane that allows passage of axons but restricts the cell body and non-neuronal cells to the upper membrane surface. Proteins extracted from the cell body and axonal preparations were separated by 7.5% SDS-PAGE (10 g/lane) and transferred to PVDF membrane. To demonstrate the purity of the DRG axonal preparation, membranes were probed with antibodies against MAP-2 and GAP43. MAP2 protein resides in the cell soma and dendrites but does not extend into the axon (37), whereas GAP43 is localized to the growing axons and soma (56). Our immunoblot showed that MAP2 was present in the cell body fraction but absent from axonal preparation, whereas GAP43 was more abundant in the axonal preparation than in the cell body fraction, thus confirming the purity of the axonal fraction. The immunoblot analysis revealed the presence of RISC proteins (p100, AGO2, FMRP, and Gemin3) in both the cell body and axonal fractions ( Fig. 5 ). Together with the immunofluorescence experiments on sciatic nerve fibers and dissociated DRG cultures, these data argue that axons possess proteins specific for RISC. The purpose of this experiment was to determine whether AGO2, FMRP, p100, and Gemin3 can form protein complexes in sciatic nerve fibers in response to treatment with siRNA against neuronal  -tubulin. The proteins were isolated from ϳ 1 cm distal and ϳ 1 cm proximal parts of the proximal stump dissected out 0.5 cm above the site of siRNA application. The control treatments included saline and nonspecific siRNA. Protein lysates were incubated with AGO2 or p100 antibodies overnight and precipitated using BSA preblocked protein A-Sepharose beads. The precipitated proteins were separated according to relative size by SDS-PAGE analysis in 5% gels, transferred to membranes, and probed with antibodies against AGO2, FMRP, p100, and Gemin3. In all treatment conditions, p100 was shown to coprecipitate with FMRP and AGO2 ( Fig. 6 , upper panel), whereas AGO2 was shown to coprecipitate with p100 and Gemin3 (Fig. 6, middle panel). As expected, expression of the multiprotein complex was more pronounced in response to treatment by siRNA, indicating that the presence of siRNA induces RISC formation (14). To investigate whether RNAi may act independently from the neuronal cell body, experiments with ligature and colchicine treatment were performed. The ligature was used to mechanically separate the sciatic nerve proximal stump from the neuronal cell bodies. Colchicine, which causes depolymerization of microtubules (57), was used to block axonal transport pharmacolog- ically without affecting the nerve capacity to conduct action potentials. Ligation or colchicine were applied for 24 h at the proximal stump of the sciatic nerve ϳ 1.5 cm above the site of the treatment with siRNAs. Experiments showed that neither the ligature nor colchicine affected RISC protein levels in the proximal stump above or below the ligature/colchicine application site. This observation indicated that RISC forms locally and independently from the neuronal cell body. To see whether Schwann cells may contribute to ...
Context 3
... aim of this experiment was to determine whether the known components of mammalian RISC (AGO2, FMRP, and p100) were present in the sciatic nerve fibers. For these experiments we chose to target neuronal  -tubulin because it is one of the abundant cytoskeletal proteins known to be synthesized locally in the axonal fibers (38). Proximal stumps of sciatic nerves transected at midthigh level were incubated with antitubulin siRNA, nonspecific control siRNA, or vehicle for 24 h. For protein extraction we used a 2 cm portion of sciatic nerve 0.5 cm above the site of treatment ( Fig. 1). For spinal cord protein extracts we used lumbar spinal cord from the corresponding animals. Protein samples isolated from sciatic nerves and spinal cords were resolved by SDS/PAGE electrophoresis and transferred to membranes, which were subsequently probed with antibodies against AGO2, FMRP, and p100. These antibodies have been successfully used elsewhere (14). Immunoblot analysis revealed the presence of AGO2, FMRP, and p100 proteins in samples from the spinal cord ( Fig. 2 , upper panel) and sciatic nerves (Fig. 2, lower panel). In sciatic nerves, pretreatment with antitubulin siRNA visibly increased the level of p100 expression, which may be a sign of induction by siRNA. The data of this experiment demonstrated that the three known components of RISC (AGO2, FMRP, and p100) were present in sciatic nerve fibers. To confirm our finding at the histological level, we performed immunofluorescence on sciatic nerve fibers to investigate the distribution of the known (AGO2, FMRP, and p100) and suspected (Gemin3) components of RISC. Longitudinal sections of an ϳ 2 cm portion of sciatic nerve proximal stump ( ϳ 0.5 cm above the site of siRNA treatment) were incubated with primary antibodies against target proteins and subsequently incubated with secondary antibodies conjugated with Texas Red, FITC, or CY3. The sections were analyzed using a Zeiss LSM 510 confocal laser scanning microscope. Triple immunostaining of these sections revealed immunoreactivity in the sciatic nerve for p100 (blue color-coded), AGO2 (red), FMRP (green), and Gemin3 (green) proteins ( Fig. 3 , upper and lower panels). A merge of the images showed coexpression of p100 (blue), AGO2 (red), Gemin3 (green) (Fig. 3, upper panel) as well as coexpression of p100 (blue), AGO2 (red), and FMRP (green) (Fig. 3, lower panel). These data demonstrated that AGO2, FMRP, p100, and Gemin3 coex- press in peripheral nerve fibers, providing the neces- sary substrates for the formation of RISC. To investigate whether RISC proteins are present in axons of dissociated neuronal cultures, we performed immunofluorescence on rat DRG neurons plated at low density according to a protocol described elsewhere (37, 38). The preparations were stained with antibodies against AGO2, p100, FMRP, and Gemin3, as well as with TUJ1 antibodies against neuronal  -tubulin and antibodies against growth-associated protein 43 (GAP43). In some experiments, DRG cultures were treated with antitubulin siRNA or nonspecific control for 24 h before immunostaining. Immunofluorescence showed colocalization of p100 with  -tubulin ( Fig. 4 A ), p100 with FMRP (Fig. 4 B ), p100 with AGO2 (Fig. 4 C ), and p100 with Gemin3 (Fig. 4 D ). A merge of the images showed a complete overlap between immunofluorescence of the proteins of interest in axons as well as in cell bodies. Incubation of DRG cultures with siRNAs demonstrated successful uptake of siRNA duplexes. FITC-conjugated siRNAs were detected in axons and bodies of DRG neurons. Triple immunofluorescence of DRG cultures treated with antitubulin siRNA confirmed colocalization of p100 with  -tubulin (Fig. 4 E ), p100 with GAP43 (Fig. 4 F ), p100 with AGO2 (Fig. 4 G ), and p100 with FMRP (Fig. 4 H ). These results clearly demonstrated the existence of RISC proteins in axons. To confirm this finding at the protein level, we performed immunoblot analysis on axonal proteins isolated from DRG cultures according to a method described previously (38). This method allows the separation of DRG processes from cell body and non- neuronal cells by culturing neurons on a porous membrane that allows passage of axons but restricts the cell body and non-neuronal cells to the upper membrane surface. Proteins extracted from the cell body and axonal preparations were separated by 7.5% SDS-PAGE (10 g/lane) and transferred to PVDF membrane. To demonstrate the purity of the DRG axonal preparation, membranes were probed with antibodies against MAP-2 and GAP43. MAP2 protein resides in the cell soma and dendrites but does not extend into the axon (37), whereas GAP43 is localized to the growing axons and soma (56). Our immunoblot showed that MAP2 was present in the cell body fraction but absent from axonal preparation, whereas GAP43 was more abundant in the axonal preparation than in the cell body fraction, thus confirming the purity of the axonal fraction. The immunoblot analysis revealed the presence of RISC proteins (p100, AGO2, FMRP, and Gemin3) in both the cell body and axonal fractions ( Fig. 5 ). Together with the immunofluorescence experiments on sciatic nerve fibers and dissociated DRG cultures, these data argue that axons possess proteins specific for RISC. The purpose of this experiment was to determine whether AGO2, FMRP, p100, and Gemin3 can form protein complexes in sciatic nerve fibers in response to treatment with siRNA against neuronal  -tubulin. The proteins were isolated from ϳ 1 cm distal and ϳ 1 cm proximal parts of the proximal stump dissected out 0.5 cm above the site of siRNA application. The control treatments included saline and nonspecific siRNA. Protein lysates were incubated with AGO2 or p100 antibodies overnight and precipitated using BSA preblocked protein A-Sepharose beads. The precipitated proteins were separated according to relative size by SDS-PAGE analysis in 5% gels, transferred to membranes, and probed with antibodies against AGO2, FMRP, p100, and Gemin3. In all treatment conditions, p100 was shown to coprecipitate with FMRP and AGO2 ( Fig. 6 , upper panel), whereas AGO2 was shown to coprecipitate with p100 and Gemin3 (Fig. 6, middle panel). As expected, expression of the multiprotein complex was more pronounced in response to treatment by siRNA, indicating that the presence of siRNA induces RISC formation (14). To investigate whether RNAi may act independently from the neuronal cell body, experiments with ligature and colchicine treatment were performed. The ligature was used to mechanically separate the sciatic nerve proximal stump from the neuronal cell bodies. Colchicine, which causes depolymerization of microtubules (57), was used to block axonal transport pharmacolog- ically without affecting the nerve capacity to conduct action potentials. Ligation or colchicine were applied for 24 h at the proximal stump of the sciatic nerve ϳ 1.5 cm above the site of the treatment with siRNAs. Experiments showed that neither the ligature nor colchicine affected RISC protein levels in the proximal stump above or below the ligature/colchicine application site. This observation indicated that RISC forms locally and independently from the neuronal cell body. To see whether Schwann cells may contribute to the expression of RISC proteins, we performed double immunostaining with antibodies ...
Similar publications
Background
Axonal regeneration depends on many factors, such as the type of injury and repair, age, distance from the cell body and distance of the denervated muscle, loss of surrounding tissue and the type of injured nerve. Experimental models use tubulisation with a silicone tube to research regenerative factors and substances to induce regenerat...
Citations
... MiRNAs can inhibit the translation of partially complementary target messe RNAs by directing the sequence-specific degradation of both fully and partially com mentary target mRNAs [85,86]. However, regarding the specific role of Gemin3 in miR biogenesis, researchers suggest that Gemin3 is a member of RISC, given the presen the Gemin3 protein in the peripheral axons of the mouse sciatic nerve and its capaci form a multiprotein RISC in response to specific treatment [72]. ...
... RISC was not previously identified as having decapping components for siRNAs miRNAs within the complex. Consequently, researchers posit that Gemin3 serves decapping enzyme within the RISC complex and plays a role in RNA decapping o combination during miRNA maturation as well as in target RNA recognition [72]. The RNase III nucleic acid endonuclease then cleaves miRNA, generating pre-miRNA, which is subsequently transported to the cytoplasm. ...
... MiRNAs can inhibit the translation of partially complementary target messenger RNAs by directing the sequence-specific degradation of both fully and partially complementary target mRNAs [85,86]. However, regarding the specific role of Gemin3 in miRNA biogenesis, researchers suggest that Gemin3 is a member of RISC, given the presence of the Gemin3 protein in the peripheral axons of the mouse sciatic nerve and its capacity to form a multiprotein RISC in response to specific treatment [72]. ...
DEAD-box decapping enzyme 20 (DDX20) is a putative RNA-decapping enzyme that can be identified by the conserved motif Asp–Glu–Ala–Asp (DEAD). Cellular processes involve numerous RNA secondary structure alterations, including translation initiation, nuclear and mitochondrial splicing, and assembly of ribosomes and spliceosomes. DDX20 reportedly plays an important role in cellular transcription and post-transcriptional modifications. On the one hand, DDX20 can interact with various transcription factors and repress the transcriptional process. On the other hand, DDX20 forms the survival motor neuron complex and participates in the assembly of snRNP, ultimately affecting the RNA splicing process. Finally, DDX20 can potentially rely on its RNA-unwinding enzyme function to participate in microRNA (miRNA) maturation and act as a component of the RNA-induced silencing complex. In addition, although DDX20 is not a key component in the innate immune system signaling pathway, it can affect the nuclear factor kappa B (NF-κB) and p53 signaling pathways. In particular, DDX20 plays different roles in tumorigenesis development through the NF-κB signaling pathway. This process is regulated by various factors such as miRNA. DDX20 can influence processes such as viral replication in cells by interacting with two proteins in Epstein–Barr virus and can regulate the replication process of several viruses through the innate immune system, indicating that DDX20 plays an important role in the innate immune system. Herein, we review the effects of DDX20 on the innate immune system and its role in transcriptional and post-transcriptional modification processes, based on which we provide an outlook on the future of DDX20 research in innate immunity and viral infections.
... In 2003, Caudy et al. reported for the first time that the Tudor-SN is a component of the RISC (RNA-induced silencing complex) [46]. Subsequently, there have been several lines of evidence that in multiple species, the Tudor-SN protein can interfere with target mRNA by participating in RISC assembly, but its calcium-dependent nuclease activity is different [9,[46][47][48][49][50][51][52][53][54]. For instance, the Tudor-SN protein in the RISC of Arabidopsis and African trypanosomes possess no or only minor nuclease activity [49][50][51][52]55]. ...
Tudor-SN (Tudor staphylococcal nuclease), also known as p100 or SND1 (Staphylococcal nuclease and Tudor domain containing 1), is a structurally conserved protein with diverse functions. Emerging evidence indicates that Tudor-SN plays an essential role in both physiological and pathological processes. Under physiological conditions, Tudor-SN regulates DNA transcription, RNA splicing, RNA stability, RNA interference, and RNA editing, and it is essential for a series of cellular biological events, such as cell cycle progression, cell metabolism, and cell survival, in response to harmful stimuli; thus, Tudor-SN functions as a “friend” to the body. However, Tudor-SN is highly expressed in most tumor cells. As an oncoprotein, Tudor-SN is closely associated with the initiation, development, and metastasis of tumors; thus, Tudor-SN functions as a “foe” to the body. What is the potential mechanism by which Tudor-SN switches from its role as “friend” to its role as “foe”? In this study, we review and summarize the available evidence regarding Tudor-SN protein structure, expression, modification, and mutation to present a novel model of Tudor-SN role switching. This review provides a comprehensive insight into the functional significance of the Tudor-SN protein under physiological and pathological conditions as well as corresponding therapeutic strategies that target Tudor-SN.
... Cellular machinery regulating the processing, abundance, and function of ncRNAs, especially miRNAs, has been shown to function in many forms of neuronal plasticity and is central to generating enduring physiological changes following nerve injury. Proteins involved in the regulation of miRNA biogenesis and RISC-mediated repression are present in the distal axons of sensory neurons and are regulated in an injuryresponsive pattern (Kim et al., 2015;Murashov et al., 2007;D. Wu et al., 2011;D. ...
Neuropathic pain is a chronic condition arising from damage to somatosensory pathways that results in pathological hypersensitivity. Persistent pain can be viewed as a consequence of maladaptive plasticity which, like most enduring forms of cellular plasticity, requires altered expression of specific gene programs. Control of gene expression at the level of protein synthesis is broadly utilized to directly modulate changes in activity and responsiveness in nociceptive pathways and provides an effective mechanism for compartmentalized regulation of the proteome in peripheral nerves through local translation. Levels of noncoding RNAs (ncRNAs) are commonly impacted by peripheral nerve injury leading to persistent pain. NcRNAs exert spatiotemporal regulation of local proteomes and affect signaling cascades supporting altered sensory responses that contribute to hyperalgesia. This review discusses ncRNAs found in the peripheral nervous system (PNS) that are dysregulated following nerve injury and the current understanding of their roles in pathophysiological pain-related responses including neuroimmune interactions, neuronal survival and axon regeneration, Schwann cell dedifferentiation and proliferation, intercellular communication, and the generation of ectopic action potentials in primary afferents. We review progress in the field beyond cataloging, with a focus on the relevant target transcripts and mechanisms underlying pain modulation by ncRNAs.
... However, we know of relatively few axonal RBPs that contribute to these mechanisms. There is also some intrinsic capacity for locally depleting specific mRNAs from axons in addition to regulating their transport, storage, and translation, since both nonsense-mediated decay (NMD) and mi-croRNA (miRNA)-stimulated RNA degradation have been shown to occur in distal axons (12)(13)(14)(15)(16). But the extent to which localized mRNAs are subjected to regulation of their decay is not known. ...
Axonally synthesized proteins support nerve regeneration through retrograde signaling and local growth mechanisms. RNA binding proteins (RBP) are needed for this and other aspects of post-transcriptional regulation of neuronal mRNAs, but only a limited number of axonal RBPs are known. We used targeted proteomics to profile RBPs in peripheral nerve axons. We detected 76 proteins with reported RNA binding activity in axoplasm, and levels of several change with axon injury and regeneration. RBPs with altered levels include KHSRP that decreases neurite outgrowth in developing CNS neurons. Axonal KHSRP levels rapidly increase after injury remaining elevated up to 28 days post axotomy. Khsrp mRNA localizes into axons and the rapid increase in axonal KHSRP is through local translation of Khsrp mRNA in axons. KHSRP can bind to mRNAs with 3’UTR AU-rich elements and targets those transcripts to the cytoplasmic exosome for degradation. KHSRP knockout mice show increased axonal levels of KHSRP target mRNAs, Gap43, Snap25, and Fubp1, following sciatic nerve injury and these mice show accelerated nerve regeneration in vivo. Together, our data indicate that axonal translation of the RNA binding protein Khsrp mRNA following nerve injury serves to promote decay of other axonal mRNAs and slow axon regeneration.
... Accordingly, the transport of molecular messengers over these long distances is highly regulated (41). Axons contain within them the complete set of protein synthesis machinery, including multiple subsets of localized mRNA that are used to synthesize proteins in response to changes in the extracellular environment and send messages to the cell body to produce a global response to stimuli (42,43). PRV hijacks these processes to promote its efficient retrograde transport from the site of infection, in the axon, to the cell body (10). ...
Infection of peripheral axons by alpha herpesviruses (AHVs) is a critical stage in establishing a life-long infection in the host. Upon entering the cytoplasm of axons, AHV nucleocapsids and associated inner-tegument proteins must engage the cellular retrograde transport machinery to promote the long-distance movement of virion components to the nucleus. The current model outlining this process is incomplete and further investigation is required to discover all viral and cellular determinants involved as well as the temporality of the events. Using a modified tri-chamber system, we have discovered a novel role of the pseudorabies virus (PRV) serine/threonine kinase, US3, in promoting efficient retrograde transport of nucleocapsids. We discovered that transporting nucleocapsids move at similar velocities both in the presence and absence of a functional US3 kinase; however fewer nucleocapsids are moving when US3 is absent and move for shorter periods of time before stopping, suggesting US3 is required for efficient nucleocapsid engagement with the retrograde transport machinery. This led to fewer nucleocapsids reaching the cell bodies to produce a productive infection 12hr later. Furthermore, US3 was responsible for the induction of local translation in axons as early as 1hpi through the stimulation of a PI3K/Akt-mToRC1 pathway. These data describe a novel role for US3 in the induction of local translation in axons during AHV infection, a critical step in transport of nucleocapsids to the cell body.
Importance
Neurons are highly polarized cells with axons that can reach centimeters in length. Communication between axons at the periphery and the distant cell body is a relatively slow process involving the active transport of chemical messengers. There’s a need for axons to respond rapidly to extracellular stimuli. Translation of repressed mRNAs present within the axon occurs to enable rapid, localized responses independently of the cell body. AHVs have evolved a way to hijack local translation in the axons to promote their transport to the nucleus. We have determined the cellular mechanism and viral components involved in the induction of axonal translation. The US3 serine/threonine kinase of PRV activates Akt-mToRC1 signaling pathways early during infection to promote axonal translation. When US3 is not present, the number of moving nucleocapsids and their processivity are reduced, suggesting that US3 activity is required for efficient engagement of nucleocapsids with the retrograde transport machinery.
... RNA binding proteins (RBP) and mRNAs assemble into a ribonucleoprotein particle (RNP) for transport into axons, and those or other RBPs can also subsequently regulate the translation of the mRNA in the axons or provide a storage depot to sequester the mRNA until needed (Dalla Costa et al., 2020). Both nonsense-mediated decay (NMD) and microRNA (miRNA)-stimulated RNA degradation have been shown to occur in distal axons (Aschrafi et al., 2012;Colak et al., 2013;Hengst et al., 2006;Murashov et al., 2007;Vargas et al., functional or domain descriptions and validated expression in the nervous system (cross-referencing each for RNA expression using the GeneCards database (https://www.genecards.org)), we arrived at 357 proteins to test. ...
Proteins generated by localized mRNA translation in axons support nerve regeneration through retrograde injury signaling and localized axon growth mechanisms. RNA binding proteins (RBP) are needed for this and other aspects of post-transcriptional control of localized mRNAs, but only a limited number of axonal RBPs have been reported. We used a targeted mass spectrometry approach to profile the axonal RBPs in naïve, injured and regenerating PNS axons. We detected 76 axonal proteins that are reported to have RNA binding activity, with the levels of several of these axonal RBPs changing with axonal injury and regeneration. These axonal RBPs with altered axoplasm levels include KHSRP that we previously reported decreases neurite outgrowth in developing CNS neurons. We show that KHSRP levels rapidly increase in sciatic nerve axons after crush injury and remain elevated increasing in levels out to 28 days post-sciatic nerve crush injury. Khsrp mRNA localizes into axons and the rapid increase in axonal KHSRP after axotomy is mediated by the local translation of its mRNA. KHSRP binds to mRNAs with a 3’UTR AU-rich element and targets those mRNAs to the cytoplasmic exosome for degradation. KHSRP knockout mice show increased axonal levels of defined KHSRP target mRNAs, Gap43 and Snap25 mRNAs, following sciatic nerve injury and accelerated nerve regeneration in vivo . These data indicate that axonal translation of Khsrp mRNA following nerve injury serves to destabilize other axonal mRNAs and slow axon regeneration.
... 19 It is also a component of the RNA-induced silencing complex and regulates the process of miRNA maturation. 20,21 In addition, DDX20 is found to modulate some transcription factors such as steroidogenic factor 1 and early growth response protein 2 through its nonconserved C-terminal domain. [22][23][24] Our study builds up a link between DAPK and DDX20 pathways, which will bring many potential research directions for both proteins. ...
Death associated Protein Kinase 1(DAPK) is a calcium/calmodulin kinase playing a vital role as a suppressor gene in various cancers. Yet its role and target gene independent of p53 is still unknown in Hepatocellular Carcinoma Cell (HCC). In this study, we discovered that DAPK suppressed HCC cell migration and invasion instead of proliferation or colony formation. Using proteomics approach, we identified DEAD‐Box Helicase 20(DDX20) as an important downstream target of DAPK in HCC cells and was critical for DAPK‐mediated inhibition of HCC cell migration and invasion. Using integrin inhibitor RGD and GTPase activity assays, we discovered that DDX20 suppressed HCC cell migration and invasion via CDC42‐integrin pathway, which was reported as an important downstream pathway of DAPK in cancer before. Further research using cycloheximide found that DAPK attenuates the proteasomal degradation of DDX20 protein, which is dependent on the kinase activity of DAPK. Our results shed light on new functions and regulation for both DAPK and DDX20 in carcinogenesis and provides new potential therapeutic targets for HCC.
... Localization and traffic of FMRP have been investigated in dendrites and postsynapses, where FMRP reportedly regulates local translation for synaptic plasticity (Huber et al., 2002;Hou et al., 2006). However, FMRP is also localized and trafficked in axons and growth cones (Antar et al., 2006;Hengst et al., 2006;Price et al., 2006;Murashov et al., 2007). Notably, these studies described alterations in axon elongation and projections in FXS model mouse neurons (Antar et al., 2006;Bureau et al., 2008), suggesting that FMRP is also important for axonal functions such as axonal outgrowth and axon guidance. ...
Fragile X mental retardation protein (FMRP) is an RNA-binding protein that regulates local translation in dendrites and spines for synaptic plasticity. In axons, FMRP is implicated in axonal extension and axon guidance. We previously demonstrated the involvement of FMRP in growth cone collapse via a translation-dependent response to Semaphorin-3A (Sema3A), a repulsive axon guidance factor. In the case of attractive axon guidance factors, RNA-binding proteins such as zipcode binding protein 1 (ZBP1) accumulate towards the stimulated side of growth cones for local translation. However, it remains unclear how Sema3A effects FMRP localization in growth cones. Here, we show that levels of FMRP in growth cones of hippocampal neurons decreased after Sema3A stimulation. This decrease in FMRP was suppressed by the ubiquitin-activating enzyme E1 enzyme inhibitor PYR-41 and proteasome inhibitor MG132, suggesting that the ubiquitin-proteasome pathway is involved in Sema3A-induced FMRP degradation in growth cones. Moreover, the E1 enzyme or proteasome inhibitor suppressed Sema3A-induced increases in microtubule-associated protein 1B (MAP1B) in growth cones, suggesting that the ubiquitin-proteasome pathway promotes local translation of MAP1B, whose translation is mediated by FMRP. These inhibitors also blocked the Sema3A-induced growth cone collapse. Collectively, our results suggest that Sema3A promotes degradation of FMRP in growth cones through the ubiquitin-proteasome pathway, leading to growth cone collapse via local translation of MAP1B. These findings reveal a new mechanism of axon guidance regulation: degradation of the translational suppressor FMRP via the ubiquitin-proteasome pathway.
... Subsequently, studies have also explored the role of miRISC composition in the regulation of the above-mentioned mechanism. Multiple components of miRISC machinery are shown to localize in developing axons and growth cones (Hengst et al., 2006;Murashov et al., 2007;Dajas-Bailador et al., 2012). A recent study has shown the co-localization of core miRISC (AGO2 and miRNAs) with mitochondria at axonal branch points and growth cone of peripheral nerve axons (Gershoni-Emek et al., 2018). ...
Activity-dependent protein synthesis plays an important role during neuronal development by fine-tuning the formation and function of neuronal circuits. Recent studies have shown that miRNAs are integral to this regulation because of their ability to control protein synthesis in a rapid, specific and potentially reversible manner. miRNA mediated regulation is a multistep process that involves inhibition of translation before degradation of targeted mRNA, which provides the possibility to store and reverse the inhibition at multiple stages. This flexibility is primarily thought to be derived from the composition of miRNA induced silencing complex (miRISC). AGO2 is likely the only obligatory component of miRISC, while multiple RBPs are shown to be associated with this core miRISC to form diverse miRISC complexes. The formation of these heterogeneous miRISC complexes is intricately regulated by various extracellular signals and cell-specific contexts. In this review, we discuss the composition of miRISC and its functions during neuronal development. Neurodevelopment is guided by both internal programs and external cues. Neuronal activity and external signals play an important role in the formation and refining of the neuronal network. miRISC composition and diversity have a critical role at distinct stages of neurodevelopment. Even though there is a good amount of literature available on the role of miRNAs mediated regulation of neuronal development, surprisingly the role of miRISC composition and its functional dynamics in neuronal development is not much discussed. In this article, we review the available literature on the heterogeneity of the neuronal miRISC composition and how this may influence translation regulation in the context of neuronal development.
... Beyond mechanisms of mRNA localization, there are many additional factors and mechanisms to regulate axonal homeostasis. The RNAi pathway ( Hengst et al., 2006;Murashov et al., 2007) and dynamic N6-methyladenosine (m6A) modification ( Yu et al., 2017) are functional in axons and regulate local translation. Local protein synthesis of Robo3.2 is regulated by axonal nonsense-mediated mRNA decay, influencing axonal pathfinding ( Colak et al., 2013). ...
The development, maturation, and maintenance of the mammalian nervous system rely on complex spatiotemporal patterns of gene expression. In neurons, this is achieved by the expression of differentially localized isoforms and specific sets of mRNA-binding proteins (mRBPs) that regulate RNA processing, mRNA trafficking, and local protein synthesis at remote sites within dendrites and axons. There is growing evidence that axons contain a specialized transcriptome and are endowed with the machinery that allows them to rapidly alter their local proteome via local translation and protein degradation. This enables axons to quickly respond to changes in their environment during development, and to facilitate axon regeneration and maintenance in adult organisms. Aside from providing autonomy to neuronal processes, local translation allows axons to send retrograde injury signals to the cell soma. In this review we discuss evidence that disturbances in mRNP transport granule assembly, axonal localization, and local translation contribute to pathology in various neurodegenerative diseases, including spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and Alzheimer's disease (AD).
